Serum-free medium and method for amplifying hematopoietic stem cells

A technology of serum-free medium and hematopoietic stem cells, applied in blood/immune system cells, culture process, tissue culture, etc., to improve the effect of expansion

Inactive Publication Date: 2019-12-13
生之宝国际再生医学科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although these techniques have provided useful media for the expansion of hematopoietic stem c

Method used

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  • Serum-free medium and method for amplifying hematopoietic stem cells
  • Serum-free medium and method for amplifying hematopoietic stem cells
  • Serum-free medium and method for amplifying hematopoietic stem cells

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0093] Experimental example 1: Evaluation of hematopoietic stem cell culture medium

[0094] To evaluate various media for in vitro expansion of hematopoietic stem cells (HSCs), IMDM containing essential cytokines, 5% cord serum (CS) and feeder layers were used as controls. Two media (SCGM and X-VIVO15) were tested in the absence of serum (but in the presence of the same cytokines and feeder layers) to see if they could be used as serum-free media. The detailed experimental steps are as follows.

[0095] On day -1, inoculate umbilical cord mesenchymal stem cells (UC-MSCs) in T-12.5 containers and culture them as feeder cells in complete culture medium (containing 10% human umbilical cord serum and DMEM) . On day 0, CD34+ hematopoietic stem cells were thawed and mixed with the above-mentioned UC-MSC feeder cells at a cell density of 2.5×10 4 Cells / ml were co-cultured for 12 days, and the following different media were used together: (1) Positive control group (PC): containin...

experiment example 2

[0099] Experimental example 2: Effect evaluation of auxiliary ingredients

[0100] This experimental example evaluates the effect of adding 4 auxiliary components to the medium containing the feeder layer. The specific experimental steps are as follows.

[0101] On day -1, inoculate umbilical cord mesenchymal stem cells (UC-MSCs) in T-12.5 containers and culture them as feeder cells in complete culture medium (containing 10% human umbilical cord serum and DMEM) . On day 0, CD34+ hematopoietic stem cells were thawed and mixed with the above-mentioned UC-MSC feeder cells at a cell density of 2.5×10 4Cells / ml were co-cultured for 12 days, and the following different media were used together: (1) positive control group (PC): IMDM containing 5% umbilical cord serum, 6 kinds of cytokines and hydrocortisone; (2) SCGM Group: SCGM containing 6 cytokines and hydrocortisone; and, (3) SCGM+SP4 group: SCGM containing 6 cytokines, hydrocortisone and 4 auxiliary components. The six cytok...

experiment example 3

[0103] Experimental example 3: Using serum-free umbilical cord mesenchymal stem cell conditioned medium to replace the feeder layer

[0104] To test potential alternatives to feeder layers, we tested the effect of serum-free umbilical cord mesenchymal stem cell conditioned medium (SF-UCM) derived from umbilical cord mesenchymal stem cells (UC-MSCs). The serum-free umbilical cord mesenchymal stem cell conditioned medium is prepared, for example, by the method mentioned above. The method comprises the steps of: (a) culturing umbilical cord mesenchymal stem cells in a serum-free cell medium (eg, serum-free SCGM); and (b) isolating the conditioned medium. The experimental results of using the umbilical cord mesenchymal stem cell conditioned medium to replace the feeder layer are as follows: Figure 3A and Figure 3B shown.

[0105] exist Figure 3A and Figure 3B Among them, the positive control group (PC) was the above-mentioned IMDM. Figure 3A and Figure 3B The PC and S...

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Abstract

The invention provides a serum-free medium and a method for amplifying hematopoietic stem cells. The serum-free culture medium comprises a serum-free basal culture medium, cytohormone, an umbilical cord mesenchymal stem cell conditioned culture medium and auxiliary components. The cytohormone comprises a stem cell factor, thrombopoietin and a hematopoietic growth factor Flt3 ligand. The umbilicalcord mesenchymal stem cell conditioned medium is derived from cultured human umbilical cord mesenchymal stem cells. The auxiliary components comprise vitamin C, vitamin E or a combination of vitamin Cand vitamin E.

Description

technical field [0001] The invention relates to a serum-free medium, in particular to a serum-free medium and a method for expanding hematopoietic stem cells. Background technique [0002] Umbilical cord blood transplantation (UBCT) is a new treatment option for previously incurable diseases. However, cord blood transplantation in adults is limited by the small number of primitive hematopoietic stem cells (HSCs) available in each transplanted individual. A small number of primitive hematopoietic stem cells can lead to delayed engraftment after transplantation. So far, attempts to expand umbilical cord blood precursor cells ex vivo have not been very successful. The ex vivo expansion approach usually results in the expansion of mature hematopoietic stem cells but not the expansion of immature hematopoietic stem cells. In addition, ex vivo expansion of umbilical cord blood hematopoietic stem cells may lead to defects such as promoting apoptosis, disrupting marrow homing, an...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2500/38C12N2500/84C12N2500/90C12N2501/125C12N2501/145C12N2501/26
Inventor 罗玮瑜曾佩娸余昭勋
Owner 生之宝国际再生医学科技股份有限公司
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